PROJECT SUMMARY/ABSTRACT Description. Our goal is to develop a multi-purpose nanotherapy tailored for osteomyelitis caused by biofilm- forming, drug-resistant bacteria that tend to reside inside osteoblasts. Osteomyelitis frequently persists or recurs despite several weeks of aggressive antibiotic therapy. Treatment failure is often caused by drug delivery related issues such as inadequate intra-bone levels of antibiotics, difficulty in eradicating biofilm-forming bacterial strains especially methicillin-resistant Staphylococcus aureus (MRSA) that require much higher therapeutic antibiotic levels, and manifestation of dose-limiting drug toxicity associated with excessive drug distribution to non-bone tissues. This set of inter-related issues can be simultaneously tackled with our bone- targeting hybrid nanocarriers (BTNs). BTNs are tailored to deliver new generation antibiotics such as linezolid and rifampin in a bone-specific and controlled manner. Our preliminary studies of the BTN prototype demonstrated that this new nanosystem could substantially bind, distribute and penetrate to various bones both in vitro and in vivo after direct exposure, local injection or systemic injection, and achieve controlled release of the linezolid payload for several days. This led to over 8-fold increase in linezolid intra-bone level versus free linezolid and significantly improved efficacy in eradicating MRSA biofilms. In this application, we will focus on further exploring this advanced nano-antibiotic therapy and enhancing its translational potential as a versatile treatment that can deal with different osteomyelitis conditions by serving as a systemic or local injectable form. Specifically, we will (1) study the parameters of linezolid-BTN for optimal treatment of drug- resistant bacteria that cause osteomyelitis, (2) determine the bone-targeting and pharmacokinetic properties of locally or systemically administered linezolid-BTNs, and (3) evaluate the therapeutic outcomes and toxicity of linezolid-BTNs in osteomyelitis models. Relevance to Public Health. Bone infection, frequently known as osteomyelitis, occurs to both children and adults especially after traumatic injuries. Currently, up to one third of post-traumatic patients face the risk of osteomyelitis. This is one of the most difficult and costly-to-treat infections as weeks or months of aggressive antibiotic therapy is required to slowly eradicate the bacteria hidden in the infected bone tissue and bone cells. Without effective treatment, osteomyelitis can easily persist and become a chronic disease and cause painful, debilitating morbidity to the patients. Successful completion of this project will validate and optimize a new nanotechnology that can substantially increase the selectivity of antibiotics for the bones, prolong their therapeutic action there and minimize the drug levels in the healthy, non-bone tissues. Consequently, a significant advance in the efficacy and safety of antibiotic treatment of this challenging disease can be achieved.